The I'eteHnaU.flmrna11997, 154, 11-34 + Review
Modes of Action of Anthelmintic Drugs
R.J. MARTIN Depa,/men/ o/ l',e,li,i,al Vete,'i,m,y .Sciences. R. CDOS.V.S., Su,n,,,e,hall. University' of Edinbu,gh, Edinburgh Etq9 I QH, UK
SUMMARY Modes of action of anthelmintic drugs are described. Some anthelmintic drugs act rapidly and selectively on neuronmscular transmission of nematodes. Levamisole, pyrantel and morantel are agonists at nicotinic acetylcholine receptors of nematode muscle and cause spastic paralysis. Dichlorvos and haloxon are organophosphorus cholinesterase antagonists. Piperazine is a GABA (T-amino-bu~ric acid) agonist at receptors on nematode muscles and causes flaccid paralysis. The avermectins increase the opening of glutanmte-gated chh)ride (GltlC1) channels and produce paralysis of phmTngeal pumping. Praziquantel has a selective efl'ect on the tegument of trematodes and increases permeability of calcium. Other anthehnintics have a biochemical mode of action. The benzimidazole drugs bind selectively to ~-tubulin of nematodes, cestodes and fluke, and inhibit nficrotubule formation. The salicylanilides: rafoxanide, oxych)zanide, brotianide and closantel and the substituted phenol, nitroxynil, are proton ionophores. Clorsuhm is a selective antagonist of fluke phosphoglycerate kinase and mutase. Diethylcarbamazine blocks host, and possibly parasite, enzymes involved in arachidonic acid metabolism, and enhances the innate, nonspecific immune system. Kx-~WOnl)S: Mode of action; anthehnintics; levamisole; dichlorvos; piperazine; avermectins; praziquantel; benzimidazoles; salicylanilides; clorsulon; diethylcarbamazine.
INTRODUCTION NICOTINIC AGONISTS
Parasitic nematodes affect animals and man caus- Levamisole, lmtamisole, pyrantel, morantel, ing considerable suffering and poor growth. oxantel, bephenium and thenium Fig. 1 shows the chemical structures of nicotinic Effective anthehnintic drugs, used to treat and control these infestations, must have selective anthelmintics. There are the imidazothiazoles toxic effects on these parasites. Unfortunately (levamisole and butamisole); the tetrahydropyrim- idines (pyrantel, morantel and oxantel); the quat- with the increased use of these compounds, anthelmintic resistance has appeared and ernatT ammonium salts (bephenium and theniuna) and tile pyrimidines (methyridine). increased in fl-equency (Prichard, 1994). If resist- These compounds act selectively as agonists at syn- ance to a particular anthehnintic has occurred, it aptic and extrasynaptic nicotinic acetTlcholine is likely that another anthelmintic with the same receptors on nematode muscle cells (Fig. 2) and mode of action will also be ineffective although produce contraction and spastic paralysis. The other anthehnintics with another mode of action, electrophysiological effects of levamisole, may still be effective. Clearly then, it is important pyrantel, morantel and oxantel have been studied to have an understanding of the mode of action of in greatest detail. anthehnintics in order to inform the selection of effective therapeutic agents. This rexqew describes modes of action of anthelmintic drugs (Table I).
1(190-0233/t)7/04(1011-24/S 12.00/0 © 1997 Bailli6re Tindall 12 THE VEH.:RIN..\RY .IOL'RNAI.. 154, I
Table I. Summary table of the modes of action of anthelmintic drugs
(;e,eric drueff name Iqxam/de,~ ,1 ,w,me (". K. & l '.&A. Trade .\'mm,.s Nicotinic agonists Icvanlisole Nilverm Illllanlisolt' Slyqllill pVlatltel Stl(ingid ii1( ii+~lll tcl P;u'alct'l I)cphcnhlili lht'llillni ( :anlll)ar nlcthvridinc
Acetylcholinesterase inhibitors 11;llOXOll Mulitwttrma dichhwvos
GABA agonist pipura/hic End.rid.
GiuCI potentiators ivt'l+lllt'Clill [V(IIIR'C al)alllcclill d~WalllCClill l)t~('tOlll;tX inoxidcciin ( :v(It'ctill niill)cinvcin l)
Calcium permeability increase praziqu,in tel l)l~mcit
[~-tubulin binding thial)cnda] Proton ionophores chls Inhibition of malate metabolism dialnphent'lhidc ( '.ol-il)an Inhibition of phosphoglycerate kinase C](II'SII](III In h'omec Super and mutase Inhibitor of arachidonic acid dicthvlcarl)amazine (:aricide,Filaricide metabolism and stimulation of innate immunity Electrophysiological effects of nicotinic ettes fl'mn Ascaris suum body muscles have shown anthelmintics in nematodes that bath application of tetramisole, the D/l. Intracelhllar recordings made with micropip- racemic mixture of levamisole, produces ntem- M()DES OF ACTION ()F ANTHELMINTIC DRU(;S 13 Butamisole Levamisole CH H 0 a / H N/H N CHa S Pyrantel Morantel Oxantel CHal CIita CI~:, N N N %) CHa Thenium CH:I Methyridine 0 -- CH 2 -- CH,2 -- N + -- CH._, I /CH 2- CH 2- O -- CH 2 CHa Bephenium CH a ~ O -- CH 2 -- CH 2 -- N + -- CH2 1 C> CHa' C? Fig. 1. Chmnical structures of nicotinic anthchnintics. brahe del)olarization, an increase in spike fl'e- inlents was: morantel=pyrantel>levamisole>acetyl- qnency and contraction (Aceves el al., 1970). choline (Harrow & Gration, 1985). Pvrantel and its analogues also produce depolariz- In addition to effects of tile anthehnintics on ation, increased spike actMtv and contl-action the membrane conductance of the muscle, when applied to Ascaris muscle (Aubr)' el al., 1970) Harrow and Gration (1985) described the conduc- suggesting that these compounds have a common tance dose-response curves for pyrantel and mor- mode of action. antel as being 'bell shaped'. The effect of this con- The two-microelectrode currelat-clamp and voh- centration-effect relationship is that the age-clamp teclmiques have been used to exalnine conductance-response increases then decreases as mttscle nlembrane conductance changes in Ascaris the concentration of the anthelmintics rises. One produced by acetylcholine, levamisole, pyrantel explanation tor this phenomena is that of open and morantel (Martin, 1982a; Harrow & Gration, channel block (Colquhoun & Sakmann, 1985) by 1985). These anthehnintics have I)een shown to the anthelmintic. Levamisole, pyrantel, morantel increase the meml)rane conductance and depolar- and oxantel are large organic cations, and could ize the membrane by opening non-selective cation enter the nicotinic ion-channel from the outside ion-channels that are permeable to both Na + and and tl-y to pass through the channel like Na + or K+ K+. Simultaneous application of acetylcholine and ions but produce the block at the narrow region pyrantel showed that both agonists acted on the of the channel, the selectivity filter. This block same nicotinic receptors, and the relative potency would be voltage-sensitive and increase with hyp- of the anthehnintics in bath application exper- erpolarization of the membrane and concen- 14 THE VETERINARY .JOURNAL 154, 1 (a) (c) ~ t~~ ~ Dorsal nelve cord ~ Syncylium eao ~'ry Arm Syncytium s cord I J 200 I.tm 1 mm Ventral nerve cord (b) Posterior Anterior Dorsal T ~ -YY Y V Y Y Y nerve .% cor DE3 Left ./ commissure J. ~kv .t ,k 2~ ,k v ., o w v v v Ventral YY YY YY YY YY YYYYY 7 nerve T cord Y g Y g 7 7 V T Fig. 2. Diagrams of tile neuronmscular organization of nematode somatic muscle. (a) Diagram of a s(mmlic muscle cell showing: the contractile spindle region; tile balloon-shaped bag that contains tile nucleus and glycogen granules; tile arm which is a process of tile muscle and reaches to one of tile nerve cords where i, divides into t]ngers Ihal I()lnl all electrically-coupled complex with the fingers of adjacent muscle cells and collectiveh' is known as the svncvtitml. The muscle cell possesses synaptic nicotinic acetylcholine receptors, and synaptic GABA receptors at tile svncvtial region and extrasynaptic acetylcholine and G,MIA receptors over tile surface of the muscle. (b) Diagram of the dorsal and ventral nerve cord showing the cell represented in a segment. All tile cell bodies of tile motor nettrones are contained in tile ventral nerve cord. Each segment has three right-hand commissures and one left-hand commissure. Each commissure is made up of one or two axons or dendrites of tile motor neurones that pass round the body to connect tile ventral and dorsal nerve cord. Within each segment there are 11 motor nero-ones that are divided into seven anatomical types (DEI, DE2, DE3: dorsal excitato~3.' motor neurones. DI: dorsal inhibitory motor neurones. VI: ventral inhibitory motor neurone. VI & V2: ventral excitatocv ntotor neurones. The remaining axons in the ventral nelwe cord are intersegmental neurones. Tile excitatory motor neurones are cholinergic and tile inhibitory motor neurones are GABAergic. (c) Diagrmn of a cross section of the body just caudal to the phau, ngeal muscle. It shows the relative locations of tile nerve cords, lateral lines, gut and muscle cells. tration of the anthelmintic. This property of pro- preparation (Robertson & Martin, 1993) using the ducing open channel block has been established patch-clamp technique. The ion-chalmel currents using single-channel recording techniques activated by low levamisole concentrations were (Robertson & Martin, 1993). shown to cart3 , cations and to have similar kinetics to acetylcholine-activated channels. The levami- Single-channel currents activated by nicotinic sole activated channels have a mean open time of anthelmintics 1.34 ms and a conductance in the range 20-45 pS. Initially, levamisole-activated channel currents At higher concentrations of levamisole, a flicker- [Fig. 3(a)] were recorded fi'om the muscle vesicle ing open channel block that increases on hyper- MODES OF ACTION OF ANTHELMINTI(" DRUGS 15 polarization is obsel-ved [Fig. 3(b)]. At -50 mV with oxantel, occupying an intermediate position the dissociation constant for the channel block between morantel and oxantel. The least potent was 123 ~,xl. In addition to the flickering block, agonist in Ascaris is oxantel which produces the clusters of openings were also observed at high lowest probabilitT of channel opening even at levamisole concentrations where openings were high concentrations (Dale & Martin, 1995). hater- separated by long (seconds) closed times: this estingly, oxantel is not effective therapeutically chlstering behaviour is similar to that asstuned to against ascariasis but is used instead to treat be desensitization in vertebrates but was more 7)'ichuris infections. The efficacy of oxantel against voltage-sensitive and more prominent at hyperpol- 7)ichuds but not against AscaHs may be due to dif- arized potentials in Ascaris. ferences in the nicotinic receptors of the two Pyrantel-activated channels [Fig. 3(c)] have also species of nematode: oxantel may be effective and been recorded using the same preparation produce opening of the TtJchuris nicotinic acetyl- (Robertson el al., 1992). Pvrantel-activated chan- choline receptor but not so effective on the Ascaris nel showed at least two distinguishable conduc- receptor. The concentrations of oxantel and tance levels: the main condnctance level was near pyrantel along the intestine may also be a factor 40 pS and the smaller conductance level was near affecting efficacies of the drugs. '22 pS, suggesting the presence of more than one type of nicotinic receptor in the membrane. Non-nicotinic or 'muscarinic' choliner~c receptor~ Again, the channels opened by pyrantel were in nematodes and resistance to anthelmintics shown to be pernmable to monovalent cations and In a number of vertebrate preparations, acetyl- channel-block occurred at hyperpolarized poten- choline is able to alter the probability of" opening tials. The channel block occurred more readily of voltage-dependent channels by acting via G-pro- with pyrantel than with levamisole as shown by the tein-coupled muscarinic receptors. An action in tact that the dissociation constant, /x]~, for levami- nematodes at acetylcholine receptors analogous sole was laigher than that of pyrantel (levamisole to vertebrate muscarinic receptors, modulating ~ at -50 ntV 123 ~txl; pyrantel K]~ at -50 naV voltage-sensitive ion channels has yet to be fully 37 btxl). reported. Tal)le II summarizes the channel I)locking There is biochemical exidence for the presence properties of levamisole and pyrantel and, in of 'muscarinic' receptors in Ascaris muscle: addition, morantel and oxantel. Some general (1) Donahue et aL (1982) have obsevved that points may be made by examining Table II. It can effects of cholinergic stimulation inchtdes be seen that all the nicotinic anthehnintics pro- increases in levels of cyclic-AMP; duce open channel-block, a form of self antagon- (2) Arevalo and Saz (1992) have observed that isnt, and that levamisole is the least potent at acetylcholine increases levels of phosphoD, l- blocking its own channel. The signiticance of the choline, 1,2-diacylglycerides and phosphatidic open channel block ntay relate to the ability of acid, and demonstrated the presence of phos- some nematodes to resist the effects of nicotinic pholipase C activitT. Donahue et al. (1982) anthehninitics: channel block may occur at such and Arevalo and Saz (1992) did not dis- low anthelmintic concentrations that normal con- tinguish between 'muscarinic' or 'nicotinic' centrations of anthelmintic are ineffective. cholinergic receptors. In Ascmis, the channel-blocking ability of Previous studies on nematodes have shown that pyrantel is greater than levamisole, but the resistance to nicotinic anthelmintics may take two greatest channel-block is produced by morantel forms: (1) The selection of genetically resistant Table II Voltage sensitivity of dissociation constants of open channel block by the nicotinic anthelmintics Membrane I¢33 l.evamisole 2KI, l5,rantel Kl~ Morantel KR Oxantel -50 mV 123 I.tXJ 37 IJ.Xl 12 p.xt 18.5 I.tM -75 mV 46 IJM 20 I.t~l 1 ~,xl 7.5 law Voltage-sensitivlty of h]~ e-fold every 20 mV e-fold eve D' 40 mV e-fbld every 22 mV e-fold ever), 29 mV /~ is the dissociation constant: the lower the concentration of this constant the more potent it is. At the concentration of the KB the channel is blocked for 50% of its open time so tile response would be reduced by a half. 16 THE VETERINARY JOURNAL. 154, 1 (a } Levamisole 15 10 0 % 5 1 pA L 0 5 10 15 20 ms 0 (b~ ...... i ...... i ...... C 1PA3~ms ~ ~ 0 (c) Pyrantel C 15- O 10- % C O 0- 0 5 10 15 20 ms Fig. 3. Patch-clamp records of silagle-channel currents activated by levamisole. The patch-clamp technique allows the activit3' of single ion-channels in the membrane of the cell to be recorded as the channel opens and closes. Small (lxl0 -v-' A) current pulses are recorded with this technique. The currents are rectangular in shape; C is the closed state and O is the open state. (a) Histogram of open-times and records of levamisole-activated single-channel currents. Cell-attached patches. Openings downward. Patch potential -75 mV; 10 laM levamisole in the patch pipette. Mean open time: 1.34 ms. (b) A flickering burst demonstrating channel block produced by 30 last levamisole at -75 mV, cell-attached patch. The comb effect of the channel current is characteristic of a drug moving into and blocking the ion-channel. (c) Histogram of open-times and records of pyrantel-activated single-channel currents. Cell-attached patch; openings downward; patch- potential -75 mV; 0.1 I.tM micromolar pyrantel in the patch-pipette. Mean open-time: 1.09 ms. The pyrantel channel open times are on average slightly shorter than those of levamisole. MODES OF ACTION OF ANTHELMINTIC DRUGS 17 mutants with modified nicotinic acetylcholine Dichlo,wos receptors on muscle (Lewis el al., 1980; Lewis et o H al., 1992). (2) Accomnaodation and recovery of CH30 \ li I C] /p--o--c~-c / parasites after long periods of exposure to the CH30 C1 anthelmintics (Lewis et al., 1980; Lewis et al., 1992), and which may be explained by nicotinic receptor desensitization. These resistant or recovered nematodes no Haloxon longer respond to the nicotinic anthehnintics, but 0 interestingly, may still respond to acetylcholine (Coles et al., 1975). One explanation for these C1CH2CH2 0 / resuhs is that in addition to tile nicotinic receptor on nematode muscle, there are other non-nic- otinic cholinergic receptors present on muscle not stimulated by the nicotinic anthelmintics. Such receptors may facilitate contraction by being CH3 coupled via G-proteins to vohage-activated channels. Fig. 4. (:heroical structure ofdichlorvos and haloxon. ORGANOPHOSPORUS CHOLINESTERASE Genetics of resislance to nicotinic anlhelmintics INHIBITORS Tile genetics of resistance to the anthelmintic levamisole has been studied in the laboratory Dichlmvos, haloxon using the small fi'ee-living soil nematode, Caenor- Compounds like dichlorvos and haloxon are habdili.~ eh,gans. Several hundred levamisole-resist- selective organophosphorus anti-cholinesterases ant alleles have been identified that were isolated (Fig. 4), and have an anthehnintic action, as well ill several screens (Lewis el al., 1980; Lewis e/ al., as all insecticidal, action. Dichlorvos and haloxon 1992). The genes responsible tbr this resistance can control insect parasites, as well as hehninth inclucled: h,v-1, unc-29 and unc-38 that encode tile parasites. The mode of action of these compounds proteins subunits that make up the nicotinic ion is to block tile action of the parasite enzyme, ace- channels of the nematode. In addition, other tylcholinesterase, leading to the excessive build up genes including: un, c-50, unc-63 and unc-74 that of tile neurotransmitter, acetylcholine. This mode are believed to be involved in receptor biosynth- of action also predisposes towards toxicity in the esis have been identified. host animal where acetylcholinesterase Only two strains of lev-1 (×21 and x61) were enzymes are also present. Because more selective dominant when crossed over with wild-types (non- combined anthehnintic+insecticidal agents resistant) (Fleming el al., 1994). The lev-1 (x21) (avermectins and milbemycin) are available, the strain only inw~lves mutation at a single amino- organophosphorus compounds are now used less acid, replacing glutanaic acid in the ion pore of frequently. the receptor ion channel (ill alpha-7 at position The existence of cholinesterase, the enzyme 237) with a positively-charged lysine. This change that breaks down acetylcholine, was first described is believed to be sufficient to change the ion chan- in Ascmis by Bueding (1952). The distribution of nel from cationic to anionic, i.e., to convert tile cholinesterase was described by Lee (1962) who receptor from an excitatory channel to all inhibi- used histochemical techniques. In the head tory one. The ~,-1 (x61) swain contains tile region of Ascmis, most of the enzyme activity is amino-acid leucine ill the pore of tile ion-channel associated with the contractile spindle region of (in alpha-7 at position 247): this point mutation is the muscle and is in the extracellular matrix. Lee expected to produce increased desensitization (1962) also described cholinesterase activity on and reduced affinity for levamisole, rendering lev- the muscle arms near their endings on the nerve anfisole less potent as an agonist. cords but not on the bag region of the muscle. The genetics of levamisole resistance in para- In C. elegans, three classes of acetylcholinester- sitic nematodes remains to be studied. ase are recognized: class A, class B, and class C, , GABA ,‘A, Although acetylcholinesterase is responsible for / ’ \ COOH ‘\ NH;? the breakdown of acehllcholine and involvecl in motor action in nematodes, it is also secretecl into the external environment in large quantities bv ‘4. .s717077 and other parasitic nematodes. The fun&on of llle secretecl aceh~lcholinesterase ma), be to reduce the effects of host acetylcholine in the intestine, perhaps clecreasing niucosal glanclulai secretion by the host. The original hypothesis of a biochemical ‘hold-fast’ effect (recluced acetylcho- line in the host intestine blocking peristalsis of the gut) has now been rqjectecl. The identification of the function of secreted cholinesterase hy para- H sitic nematodes, ancl the abilitv to antagonize this enz\me, mav lead to the increased use of‘ anti- cholinesterases in the future to facilitate renio\xl Piperazine of gut nematodes. 2 pA J 100ms GABA AGONIST Piperazine has a heteroq~lic ring structure (Fig. .5), ancl unlike y-amino-butyric acid (GABA), lacks a carboxvl group. Howe\*er. these t~vo con~pouncls act on th’e same receptor that is a ligancl-galecl Cl- channel found on the synaptic ancl extrasynaptic membrane of nematocle muscle (Martin, 1980; h4artin. 1982). Bath application of GABA or pip- erazine increases the opening of the muscle mem- brme <:I- channels, hvperpolarizes the membrane potential, increases tile membrane conductance and produces spastic paralysis. Piperazine appears to ha\pe a selective effect on large nematocles of and are known to be products of three separate the gastrointestinal tract, perhaps because the genes: owl, uw2 cind are-3 (Opperman & Chang, high CO, environment allows CO, to mimic the 1992). The three classes of acetylcholinesterase missing carboxy] group of piperzine. Piperazine are sepal-able by their solubility in Triton, their appears less active against nematodes that occup! temperature stability and sensitivity to cholinester- a high 0, environment. ase antagonists. In C. elegcr??.r,the major form is Piperazine- and GABA-activated channel cur- class B which is distributed in the head and body; rents (Fig. 5) ha\re been recorded using cell- classes A and C have a distribution biased towards attachecl and isolated outside-out patches (Martin, the head. Extraction of the different classes for 1985). Although the channels may have more biochemical characterization has produced evi- than one conductance level, the most frequently dence that the enzyme may exist as a monomer, observed is 22 pS for both agonists. However, the dimer and tetramer. Defects in the ace-1, m-e-2 mcl mean open time of the channels for piperazine ace-j genes suggest that the functions of the diff- was shorter, 14 ms, than that produced by GABA, erent classes are supplementary but that if there which produced channels that had mean open are defects in all of the genes, then elevated levels times of 32 ms. GABA is more than lo-100 times of acetylcholine occur in C. eleguns and there is more potent than piperazine when conductance motor incoordination (Opperman & Chang, dose-response relationships are examined cluring 1992). bath application of the agonists (Martin, 1985). MODES OF ACTION OF ANTHELMINTI(" DRUGS 19 Ivermectin The difference in potency may be explained by the fact that higher concentrations of piperazine HO~ a ~OCH'~ are required to produce the same opening rate of H3C O 0...1~ ~ CHa A .-CHa the channel as that produced by GABA, and that the average duration of the channel openings pro- duced by piperazine is much shorter. The same IlL o_._A probability of channel opening can be achieved h-o j. with lower concentrations of GABA than pipera- Abamectin ~" "~ zine. Therapeutically, however, GABA would be OCH.~ 0 ~ CH ineffective because it is not selective like pipera- HO_ 2-. hl :' zine and is highly ionized and does not cross the "~ OCHa OH cuticle. HaC/k" 0''~ 0~['~ " CH, CHaR GLUTAMATE-GATED CHLORIDE (GLUCL) l! o._.'o RECEPTOR POTENTIATORS Doramectin ; ]__ I~ Ivermectin, abamectin, doramectin, milbemycin D, moxidectin HO.~ ~.,.CHa OH The avermectins (Fig. 6) are a group of broad- spectruna, macrocyclic, lactone antibiotic anthel- mintics used to control nematode parasites in man and animals (Campbell & Benz, 1984), and assumed to have the same mode of action in both (Shoop et al., 1995). They are used to control onchocerciasis (river blindness) in humans and gastrointestinal, cardiac and respirato~-y nematode 0 HI-..~ CH:, parasites of domestic animals. The mode of action OH of the avermectins is to selectively paralyse the Milbemyein D parasite by increasing muscle CI- permeability, but the identity of the channel targeted by the aver- CHa H A ~CHa mectins has been controversial, see Arena (1994) for a recent review. Cloning of a GluCl-otl and a GIu('I-~3 subunit fi'om the model soil nematode C H3C" "]1 [ CHa elegans, and co-expression of the subunits in Xen- opus oocytes, has led to the identification of an avermectin-sensitive GIuC1 ion-channel (Cully et al., 1994). The effects of avermectins, at low con- I OH centrations, are to potentiate the effect of gluta- mate, and at higher concentrations, the avermec- Moxidectin tins open the glutanmte-gated channel directly. The selective therapeutic effects of the avennec- tins could be explained by an action on a Glu CI- c_Y Fig. 6. Chmnical structure of avermectin and milbenlycin anthelmintics. Ivermectin is at least 80% 22,23 dihydroavermectin Bla (R is sec butyl) and not 0 ~CHa more than 20% 22,23 dihydroavermectin Blb (R is isopropyl). Abamectin is at least 80% avermectin Bla (R is sec butTI) and not more than 20% avermectin Blb (R is isopropyl). Fig. 6. FI LI/,q.~.fiIIBL. Z~t)le, C 20 THE VETERINARY JOURNAL, 154, 1 Terminal i Isthmus : Carpus bulb : Metacarpus : Procarpus --- ::::::::::::::::::::::: i i::ii!iiiiiiii::ii i! i ~pmo ::.::::.: pm3 • :: pm4 • : pm3 :.::::.: ~2 E~ Fig. 7. Diagram of the phalyngeal muscle of (J. eh'gan,~ showing tile location ~f the GIuC1 receptors ~m the pro4 muscle ceils that are innervated by the m3 motor neurones. Also illustrated is the division o1 the phal)ngeal muscle into Ihe regions: lerminal bulb: isthmus: carpus (sul)divided into metacarpus and procarpus). The olhel lll(ll(}l" Ilelll'(}lles (Ill l, m2, m4 ~" mS) that innervate the muscle cells (pro3, proS, pro6, pro7) are shown. The interneurones 15 thai inhibits m3, as well as m4, is shown. Adapted tiom Avery (I ~).t)~:~). I V channel is exl)ressed ill tile I)halo'nx of C. e/egans pM4 muscle (Fig. 7) (Laughton el al., 1995). The location of the Glu(]l-0tl suhui-lit is not known. The phai)'ngeal muscle is required ti)r feeding E and is known to receive an inhihitorv motor nett- tone, M3 (Fig. 7), that is not likely to be GABA- z ergic (Laughton et al., 1995) but glutamatergic (Avel)', 1993). The location of an avermectin-sen- sitive (;luC1 channel, has heen identilied using a two microelectrode current clamp technique (Martin, 1996) in the parasitic nematode, A. Mtttm (Fig. 8). Experiments show that the pharyngeal muscle of this parasite possesses glutamate recep- tors that gate chloride channels and that are sensi- tive to the avermectin analogue milhemycin D. Head Pharynx Nerve cord Intestine Fig. 8. Diagram of the preparation used to record the Effect of glutamate electrophysiological effect of glutamate and milbemycin Fig. 9 shows effects of the application of mil- D on the phaiTngeal membrane potential and input bemycin D and glutamate oll Ascaris phal3,ngeal conductance. Two micropipettes were inserted into the muscle input conductance and membrane pharyngeal muscle: one was used to record membrane poten- potential (V); the other was used to inject current (I). tial: glutamate [Fig. 9(b)] produces a transient small hyperpolarization of 1 mV associated with an increase in membrane conductance. The effect ion-channel that is present in parasitic nematodes of glutamate but not GABA [Fig. 9(c)] in pharyn- but not present in the host animal. geal AscaTis preparations is to produce a reversible Molecular experiments using the lacZ marker increase in input conductance associated with a suggest that the GluC1-]3 subunit of the glutamate small change in membrane potential and to MODES OF ACTION OF ANTHELMINTIC DRUGS 21 (a) Effect of milbemycin D The effect of ivermectin and milbemycin D, on r" -7- .... --- ..... -- 5 mV __ the C. elegans avermectin-sensitive glutamate receptor expressed in Xenopus oocytes is to pro- iillI[llililliitlllll llll " 30s duce a potentiation of glutamate effects and to 890 nM Milbemycin produce a slow irreversible increase in conduc- tance of the membrane (Cully et al., 1994). The effects of milbemycin D on the input conductance (b) of the Ascaris pharyngeal preparation is to pro- ..... :,,,ii',iiii,,.,?d.Tifflntitl'tl[lillllllllillilil duce a small change in membrane conductance and to potentiate the effect of low concentrations of glutamate [Figs. (9a), 10(a), (b), (c) and (d)]. The location of the GIuC1 receptor or the aver- mectin binding 0t-subunit in the nematode remains to be explored fully. Recent molecular biological experiments with C. elegans on an iver- 100 laM Glut. mectin-resistant strain (avr-15) suggest that it is a GluCl-0t2 subunit that is present in the pharyngeal (c) muscle of C. elegans not the GluCl-otl (review: Cully el al., 1996). The location and function of the original GluCl-cd subunit remains to be deter- llllllIHltli! I,[!iIL!!I ll Tiii, Nl!lI,E!ll! l ! ilt mined. However, the presence of more than one 5mVl__ GluCl-ot subunit indicates the presence of more 30 s than one site of action for the avermectins. It ti lh lil ttt t tttlillhlll,Ll,li&L,ll[tl[,tlktdlt[tilhliltttt implies that the appearance of resistance to aver- mectins requires mutation of more than one 300 ~IM GABA gene. In addition to effects on the pharyngeal muscle, ivermectin also has an effect on somatic muscle and opens non-GABA activated channels, and in addition, inhibits GABA-activated channels Fig. 9. (a) Effect of 890rim milbemycin D on the memb,ane potential and input conductance. Milbemycin (Holden-Dye & Walker, 1990). Thus again it slowly p,oduccd an increase in input conductance that appears that the avermectins may have more than was not reversed on washing (not shown). (b) Effect of one site of action in parasitic nematodes. application of 100 ItM t.-glutamate on membrane potential and input conductance. Different preparation fi'om (a). Glutamate (applied during horizontal bar) produced a transient small hyperpolarization of 1 mV associated with INCREASED CALCIUM PERMEABILITY an input conductance change from 157 ItS to a peak of 429 ItS (AG: 272 ItS) desensitizing to 231 gS ( AG: 74 ItS) Praziquantel after 4 rain. (c) After washing the preparation the input Fig. 11 shows the chemical su-ucture of pra- conductance of the pharynx returned towards control ziquantel. Praziquantel has a selective toxic effect levels (142 ItS) and the effect of 300 gM GABA was tested on schistosome parasites, where its mode of action without effect (applied during the horizontal bar). The lack of effect of GABA was not due to desensitization has been studied more extensively (Andrews et al., because subsequent application of 100~xt L-glutamate 1983; Harnett, 1988) than in cestodes. Many increased the input conductance again (not shown). actions of praziquantel may be explained by an increased Ca `'+ permeability of parasite muscle inhibit pharyngeal pumping that is part of the and/or tegumental membranes. Fig. 12 is a dia- nematode parasite feeding process. Zero-C1- gram of the schistosome tegument and underlying solutions were shown to abolish reversibly the glut- muscle that illustrates the known sites where Ca `'+ amate-induced conductance responses (Martin, crosses into and between intracellular compart- 1996) and were used to demonstrate that the glut- ments, and therefore, the possible sites for pra- amate effect was mediated by a CI- channels in ziquantel action. Ascam. '22 ]'HE VETERINARVJOURNAI~, 154. 1 12°f (a) / 100 ~- 60- 4O / 2O I I I l ~ I 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Milbemycin (prq) (b) (c) (d) 5 mV 30 JaM Glut. 30 p.xl Glut. 30 p.~ Glut. 1 rain in 30 nM Milb. in 300 nM Milb. Fig. 10. (;luta,mlte potentiation effects ()f milbcmvcin D. (a) (|) Pl()l of peak A(; produced by. 30 p_~l ghmm)aw againsl milbemvcin co,lcentnuioq: (O) plot ()f resting input conductance ()f the phalynx agains! mill)cmvcin c().ccn,alion. Resuhs fiom the experiment illustrated in (b), (c) & (d).(b) ('ontrol 30 ~.txl glutamate vcsp()nsc, peak A(;, is small. 16 ~.tS. (c) In the pvescace of 30 .xt milbemvcin (D) the" peak A(; produced by 30 ~Xl gltmtmatc i.cvcased t() 27 l.tS. (d) 300 nxl milbemvcin D increased the peak A(; to 86 laS. I. this pavticttlar prel)aration the gltmlmatt" resp().se was a small depolarizing potential, indicating that the (:l-reversal p()te.tial was slightly depolarized relative, to the' mcmbra.c potential. Effect o'n Ca "-'+ permeability Praziquantel Praziquantel is known to increase ''Ca '-'+ influx across the schistosome tegument and to cause rapid muscle contraction of the parasite (Mehlhorn el al., 1981). It is known that bathing the parasites in Ca'-'+-fi'ee mediuna blocks the pra- .N ziquantel induced contraction (Fettever el al., 1980; Wolde-Mussie et al., 1982; Thompson el al., 1984) but the effect of bathing the preparation in Ca"+-fi'ee bath solution is not immediate, requiring £\ more that 10 rain to be effective. The effect of pra- ziquantel on Ca `-'+ influx suggests that the sites of action are Ca '-'+ permeable ion channels in the membrane of the tegument and muscle cell (Blair et aL, 1994). The contractions in shistosomes are Fig. 11. Chemical structure of praziqtmn tel. reversed if praziquantel is removed and may be MODES OF ACTION OF ANTHELMINTIC DRUGS 23 Double Host (Blood) membrane ua- 2+ Ca 2+ Ca 2+ 5 \ 1 ,-, I ,-, "~ Ca2+ - 2+ - Ca2+ - • z, Ida 4 b I 'V., . • " . " " - " • • V ' "Na + • _". . 7 " • • •.. • ' Fig. 12. Diagram of 1he structure of the body wall of Schi.~losoma ma,soni showing the possible sites of action of praziquantel and meclaanisms of Ca'-" transport into and out ot the tegument. 1: Vohage-activated Ca +-'+ channel. 2: II'Jll'~lct'lhll~tl" i]lessell~el activated Ca +-'" chanllel. 3: Extracelhdar receptor operated Ca ~" channel. 4: Non-selective cation channel also allowing t+ntz-v of Ca'-". 5: Na'/Ca e+ exchanger. 6: Ca `'+ ATPase pt, mping out ('J*. 7: Intrategtmlental Ca e+ buffers. 8: IP:~ releasable store from the sarcol)lasmic rcticuhtm. 9: Ca*-'" induced Ca'-" release channel (CICR channel). I0: (;~(-'" ATPase pttmp: savcoplasmic endoplasmic retictdum Ca'-'- (SERCA). 11: Electrical jtmction between mttscle cell and tegumetlt. 12: Electrical junctions between tnuscle cells. If praziqttantel acts like caffeine it would act on site 9. 13: A proton I)tttnp may set tq) a pH gradient actress the tegument and be required for qormal titnction. A proton ionophore wc+uld ttl)Set tiffs gradient and lead to the demise c~t+ the organism. blocked by Mg '-'+, or La :~+ but not I)y Ni '-'+ Co e+ 01- will eliminate the effect of praziquantel on the the calcium-channel blocker D-600 (Fetterer et al., muscle preparation in Ca'-'+-free solutions. These 1980; Wolde-Mussie et al., 1982). The application experiments suggest that caffeine and praziquan- of a high K+ solution to depolarize the schisto- tel share the same site of action. some preparation results in a rapid depolariz- In vertebrate smooth muscle preparations, caf- ation, but the application of praziquantel resultsin feine acts to stimulate the opening of a large cat- a slower onset depolarization. From these observa- ion channel known as the CICR channel (calcium- tions, it may be concluded that praziquantel some- induced calcium release channel) (Herrmann- how results in an increase in Ca `-'+ permeability Frank et al., 1991) in the sarcoplasmic reticulum. across parasite membranes via channels that are This channel is activated by cytosolic rises in Ca '-'+, not activated by depolarization. These channels adenosine triphosphate (ATP) and caffeine but is must be pharmacologically different fi'om those of inhibited by Mg '-'+. The CICR channel produces the host animal or the praziquantel would affect regenerative Ca `-'+ release from the sarcoplasmic the host animal. reticulum following entry of Ca `-'+ through voltage- Experiments on a snail smooth muscle prep- activated channels (Gregoire et al., 1989). Large aration (Gardner & Brezden, 1984) have illus- cation channels, which might be similar to the trated that caffeine mimics the effect of pra- CICR channel, have been observed in the tegu- ziquantel in producing contraction, and that prior mental membrane of schistosomes (Day et al., treatment of the muscle preparation with caffeine 1992). 24 THE VETERINARY .]OURNAL. 154, I Parasite antigen exposure secreta D, vesicles, a decreased glucose uptake and It has also been found that curative doses of an increased utilization of stored glycogen. In praziquantel in infected animals resuhs in Ascaris, mebendazole is taken up I)y phaiTngeal antigens of schistosomes being exposed and and intestinal cells where it is tound in the cyto- binding and penetration of host antigen cells to plasnaic fi'action bound to proteins with molecular the parasite after 17h treatment (Harnett & weights of about 50 and 100 kDa that represent Knsel, 1986; Brindley & Sher, 1987). The immune mollonlel-s and dimers of tubulin. Otlr current system of the host, therefore, plays an important understanding of the mode of action of the benzi- role in the praziquantel-induced death of the midazoles thus started when it was recognized that parasite. One explanation for the exposure of the the effect of mebendazole on Ascads was to dis- parasite antigen may be that increased cvtosolic rupt the microtubules of the intestinal cells pro- Ca `-'+ causes phospholipase C activation and then ducing an inability to take up ghtcose (Van den activation of protein kinase C (Berridge & h-vine, Bossche, 1972; Van den Bossche el al., 1982). It 1984; Sigiya & Furuyanaa, 1990). The phosphoDq- was found that benzimidazole anthelmintic com- ation of proteins by protein kinase C could then peted with the binding site for [H :~] colchicine on destabilize the tegument causing the observed 13-tuhulin and that potency of the I)enzimidazole vacuolation and antigen exposure (Wiest el al., antlmhnintics correlated with the dissociation con- 1992). Once again the mode of action of pra- stant for hinding to nematode I]-mbulin (Lacey, ziquantel relates to an effect on Ca `-'+ permeability 1990; Sangster et al., 1985). of the tegumental nmmhranes. Praziquantel is Micrombules are intracellular organelles that used fi'equently to treat tapeworm infestations but serve a variety of fimctions including movement of less is known of the mode of action in this group chromosomes during cell division; providing the of parasites. It is presumed that there is a common structural skeleton to the cell; movement of intra- mode of action against trematodes and cestodes. cellular particles including ener~ naetaholites: and exocvtosis. They are lound in both animal, INHIBITION OF MICROTUBULE plant, fungi and some bacterial cells (SnTer, FORMATION, ~-TUBULIN BINDING 1995). Microtul3ules are composed of two 450 amino- Benzimidazoles: thiabendazole, cambendazole, acid proteins known as c~-tubulin and I]-tt!bulin. mebendazole, fenbendazole, oxibe~Mazole, Fig. 14 is a diagrammatic representation of [3-tuhtt- oxfendazole, albendazole, albendazo& sulphoxide, lin. It is composed of three domains: domain 1 is parbendazole, flubendazole, t~4clabendazole & pro- 34 kDa in size; domain 2 is 19 kDa in size and the drugs: netobimin, febantel, thiophanate tail is 2 kDa in size. There are GTP binding sites Fig. 13 illustrates the chemical structures of the present which bind to sites I, II, III, and IV on the benzimidazole anthehnintics and prodrugs. Pro- I]-tubulila. The 13-tul)ulin is folded so that the GTP- drugs are converted to active benzimidazoles by hinding sites form a pocket. metabolic processes in the host animal so that it is the active metabolites that are responsible for the Formation of microtubules anthelmintic action. Triclahendazole, the flukici- The formation of mio'otubnles is a dynamic dal compound, is assumed to act hy the same process (Fig. 14.) Formation involves the polynmr- mechanism as the other benzimidazoles but a ization of tubulin at one end (the positive pole) clear explanation for its selective effect against and the depolymerization at tile other end (the fluke is not known (McKellar & Kinabo, 1991) but negative pole). The microtubules that tbrm are may be related to its high level of plasma protein made up of 13 tubulin molecule rings (6 0f binding. tubulin+7 13-tubulin alternating with 7 0t-tubulin+6 [3-tubulin rings). Seventy-five to 85% of the mass fl-tulmlin binding of microtubules is composed of the tubtdin pro- Mebendazole (Borgers et al., 1975; Van den teins but in addition, microtubule-associated pro- Bossche & De Nollin, 1973) and flubendazole teins (MAPs) are present stabilizing the structure. (Van den Bossche & De Nollin, 1973) induce the A number of factors favour polymerization includ- loss of cytoplasmic microtubules of the tegu- ing GTP, Mg'-'÷, and an increase in temperature (to mental and intestinal cells of cestodes and nema- 37°C). A decrease in temperature (to 4°C), the todes, and this is followed by loss of transport of presence of Ca `-'+ or cahnodulin will favour depoly- MODES OF ACTION OF ANTHELMINTIC DRUGS 25 BENZIMIDAZOLES PRODRUG NHCO2CH3 l "I N N~C NHCO2CH3 N iL S NHCOCH2OCH3 H Febantel: converts to fenbendazole Thiabendazole: R = H-- Cambendazole: R = (CHa)2CHOCONH-- BENZIMIDAZOLE CARBAMATES TRICLABENDAZOLE CI CI R .N. [ i NHCO2CH a ' ' I "~ SCH 3 N CH 3 " N H H Oxibendazole: R = CH~CHzCH20-- Albendazole: R = CHaCH2CH2S-- Fenbendazole: R = (/,, -- ~'~ S-- O Oxfendazole: R = @ S--- O El Mebendazole: R = @ C m 0 Flubendazole: R = F @,S m Fig. 13. Chemical structure of benzinfid~oles, benzimidazole carbamates, prodrug febantel and the antifluke drug triclabendazole. merization. Interestingly, c~- and [3-tubulin will the mitotic inhibitors and benzimidazoles can do pol),merize into a number of shapes (rings and tiffs by binding to ~-tubulin molecules. sheets) in addition to microtubules when in vitro Thus the mode of action of the benzimidazole preparations are made. The tbrmation of micro- anthelmintics is the selective binding to nematode tubules may be inhibited by substances that bind ~-tubulin, and consequent inhibition of micro- to the leading edge (the positive pole) of polymer- tubule formation. The effects are, therefore, ization. This process of inhibition is known as slower in onset than the anthelmintics that act as 'capping', and colchicine, vinblastine, vincristine, neurotransmitter agonists, and include slow onset 26 THE VETERINARYJOURNAL. 154, 1. [3-Tubulin Domain I: 34 KDa Domain: II 19 KDa Tail: 2 KDa 100 200IH TI 300im 400i i Hinge Microtubule formation from cx- and ~-tubulin Q) a-tubulin ~(~ 13 membered + ~ ~D~: spiral made of • ~-tubulin alternating ~- and [3-tubulin ~ Vesicle transport along Negative pole. Positive pole. Microtubule Microtubule breakdown formation increased by fall in increased temperature and Ca ++. by GTP, Mg+÷, temperature inhibited by benzimidazoles. Fig. 14. Diagram of the 50 kDa ~-tubulin protein. It consists of three domains that are separated by protease action. In the middle in a 'hinge' allowing the molecule to fold. The location of the amino acids 100, 200, 300 and 400 are shown. Below this is a diagram representing the formation of microtubules by 0t- and ~-tubulin that polymerizes by forming a 13 membered hollow spiral. Each turn of the spiral allows the 0t- and ~-tubulin to pack ahernately along the length of the microtubule. Polymerization takes place at the positive pole where temperature and GTP Mg ''÷ and other factors favour microtubule formation. The formation of microtubules is inhibited by binding of benzimidazoles to ~-tubulin to produce 'Capping' and inhibition of further microtubule formation. Breakdown occurs at the negative pole. The physiological function of the microtubules include intracellular transport via special proteins, kinesins or dynesins (here represented by the match-stick man). starvation of the nematode (intestinal cell have separate genes (Guenette et aL, 1991; disruption) and an inhibition of egg production. Guenette et aL, 1992; Lubega et al., 1994). Each of these isotypes have alleles: up to six for isotype I; Genesfor fl-tubulin and resistance and up to 12 for isotype II. It is not known if the In nematodes, two isotypes of [3-tubulin have specific isotypes have different functions in the been identified: isotype I and isotype II which nematodes. MODES OF ACTION OF ANTHEI.MINTIC DRUGS 27 A reduction in the nunfl)er of isotype alleles fi)r series of protein complexes on the inner mito- ]3-mbulin is associated with the appearance of chondrial mmnbrane. This process leads to pro- benzimidazole resistance (Roos e/ al., 1995). tons being pumped out of the mitochondria Resistance is associated with a progressive loss of matrix producing a proton motive force that is alleles of isotype 1 and a total loss of isot?'pe 2 due to the pH gradient and transmembrane elec- fi'om the nematode population. A resistant popu- tric potential. ATP is synthesized when the pro- lation of Haemonchus co~tlorlus has been charac- tons flow back into the mitochondrial matrix terized by only the presence of a single [3-tubulin through an enzyme complex. This process of oxi- allele referred to as allele 200. The appearance of dative phosphoD, lation takes place in the host ani- resistance can be explained by a loss of susceptible mal (StD, er, 1995), as well as in the parasitic hel- phenotypes and the st, rvival of a resistant pheno- minths (Van den Bossche, 1972; McKellar & type and its increased representation in the Kinabo, 1991). remaining population. Lipid soluble substances that can car D ' protons In fungi, benzimidazole resistance is associated may shuttle across the inner membrane of the with the appearance of a different form of [3-tubu- mitochondria, and remove the proton gradient lin (Fujinmra el al., 1992; Jung el al., 1992) which and uncouple oxidative phosphorylation so that is characterized by the appearance of tvrosine the oxidation of NADH and FADH is no longer instead of phenylalanine in position 200. Because linked to the production of ATP and may carry, on mammalian [3-tubulins have also tvrosine present without its production. Fig. 15 shows the position in the 200 amino acid position (Lewis el al., 1985) of the proton that is able to dissociate froni a var- it is unlikely that benzimidazole resistance may be iety of compounds. 2,4-Dilfitrophenol, carbonyl-p- overcome by changes in drug chemistvv. It would trifluoro-methoxyphenylhydrazone, (Stryer, 1995) not be possible to design a selectively-toxic agent and laexachloroplaene, nitroxynil, oxvclozanide against the ['ungi because the fungi and host ~-tub- (Corbett & Goose, 1971; Edwards el al., 1981b) are ulins would both bind the benzimidazole to the all lipophilic compounds that are capable of same degree and st) be loxic to both species. A carr}.'ing protons across membranes, and there- similar i)helmmenon may occur with nematode fore, may act to uncouple oxidative pbosphoD,1- pm-asites. ation preventing the production of the proton gradient across the inner mitochondrial membrane. PROTON IONOPHORES: SALICYLANILIDES The nleclaallism of action of the proton ionoph- AND SUBSTITUTED PHENOLS. ores has been assnmed to be to selectively uncouple oxidative pbosphoD'lation in parasite Salic~,lanilides: closantel, raJbxanide, o.~yclozanide, mitochondria. Pax and Bennett (1989) have bro/i'anide a~d substituted phenols: nitroxynil, experimental evidence that the inner membrane niclopholan, hexachorophene dibromsalan & of the parasite may not be the site of action of the niclosa mide proton iollophores. Thev observed that appli- The plaarmacolog,3, of a range of anti-fluke cation of closantel to Schistosoma mansoni and Fasci- drugs have been reviewed (McKellar & I Oxyclozanide C1 OH OH C1 CI CI CI Rafoxanide I OH C1 Fig. 16. Diagram of the mitochondria illustrating the ~CO!~ ~O~Cl inhibitory effects of an oxidative phosphorylase uncoupler on normal ATP production that is driven by the proton gradient inside the mitochondria and produced by I OH C1 tricarboxylic acid metabolism. Closantel This is explained by their strong plasma protein I OH C1 binding which is more than 99% for the salicylani- lides (Mohammed-Ali & Bogan, 1987) and 98% CON__// \/x_._?__< / \~---Cl for nitroxynil (Alvinerie et al., 1995). The selective anthehnintic action of these highly protein-bound anthelmintics may be explained, in part, by their I OH CI effect against blood-sucking parasites, concentrat- ing the anthelmintic in the parasite without the high tissue levels being produced in the host. The Brotianide high level of protein binding may explain the selective effect of these agents, and the fact that C1 CI OH well bled out carcasses have low tissue residue levels (McKellar & Kinabo, 1991). Thus the mode of action of this group of anthelmintic involves CS o< r the selective delivery of the proton ionophores to the parasite because of the high level of plasma- Br OCOCH3 CI protein binding. Nitroxynil DIAMPHENETHIDE I Diamphenethide (Fig. 17) is more active against immature Fasciola hepatica in the liver than the adult Fasciola in the bile ducts (Kendall & Parfitt, 1973). Diamphenethide is a prodrug that is deace- tylated in the host's liver to an active form which is the monoamine and the diamine (Coles, 1976). O2N The effects of the diamphenethide amine (active Fig. 15. Chemical structure of the salicylanilides form) on Fasciola in vitro have been studied by oxyclozanide, rafoxanide & closantel and the DNP Edwards et al. (1981a). These authors described derivative nitroxynil. The location of the dissociating how diamphenethide amine produces an elev- proton (H) is shown in the shaded box. ation of malate concentrations, an intermediary breakdown product of glucose metabolism. They were not able to identify an action of diamphene- The anthelmintics that have the longest half-life thide on a particular enzyme in the glycolytic in the body are the salicylanides and nitroxynil. pathway but suggested that the effect on malate MODES OF ACTION OF ANTHELMINTI(" DRU(_;S 29 Diamphenethide ./--- ,,' / \ CH3CONH -- / .... 'i:, OCHzCHzOCH2CHeO /4/, ' NHCOCH 3 / .. / Clorsulon C1 CI2 = C NH2 ~\ / H2NO2S SONH 2 Diethylcarbamazine C9H5 / \ CHaN NCON \ / ...., C2Hs Fig. 17. Chemical structure of diamphenethide, clorsuhm and diethylcarbamazine. was likely to be a prima D, effect because it plmsphoglyce,'ate kinase (Schuhnan et al., 1982) occurred early on and before the deterioration of and phosphoglyceromutase (Schulman & the whole parasite. Interestingly, in the second Valentino, 1982) of Fasciola and inhibits the paper of Edwards et al. (1981h) the effect of dopa- Emden-Meyerhoff pathways in fluke. As a result, mine, a putative neurotransmitter in Fasciola had a there is a selective inhibition of glucose utiliz- protective effect against diamphenethide. ation, and acetate and propionate formation. The The action of diamphenethide remains to be inhibition of the Fasciola phosphoglycerate kinase defined in greater detail but the study of Edwards is competitive (Fig. 18) with a ~ of 0.29 mM: clor- el aL (1981a) showed that its biochenaical effects sulon competitively inhibiting the binding of ATP contrasted with the proton ionophore, oxyclozan- and 3-phosphoglycerate to the phosphoglycerate ide and appears to involve effects on malate kinase. Schulman et al. (1982) suggested that the metabolism in Fasciola. large group on the six position of clorsulon pre- vented the conformational change in the kinase enzyme required for activity. There was also a INHIBITION OF PHOSPHOGLYCERATE good correlation between the I~ values.for antag- KINASE AND MUTASE onism of Fasciola phosphoglycerate kinase for a range of compounds related to clorsulon and the Clorsulon potency of these compounds as antifluke agents. Clorsulon is 4-amino-6 trichloro ethenyl 1,3- This evidence supports the suggested mode of benzenedisulphonamide (Fig. 17). Structurally, it action. is similar to 1,3-diphosphoglycerate (Schulman et The inhibition of Fasciola phosphoglyceromut- al., 1982) and consequently, inhibits the enzymes ase by clorsulon was studied by Schulman and 31) THE VETERINARY JOURNAl., 15-1, I E-ATP / \ E1 -- I + E 3PG-E-ATP -- ADP-E-1,3DiPG "~-----~E-3DiPG + ADP ~-~- E + 1,3DiPG / \ / " ,/ 3PG-E Fig. 18. Diagram of the competitive antagonism of 19h¢lsl)hoglyctwate kiqase (El of l,'asciola hepatica by clorsulon (1). DiphosphoiT1 glycerale: DiPG. 3-Plmsphol3l glyceratc: 3PC;. Valentino (1982) who pointed out that this does not appear to be involved because nucle, enzyme requires 2,3-diplaosphoglycerate for acti- athvmic mice, infected with BruKia pahanffi, vation and the ch)se similarity of clorsuh)n to showed a marked reduction in microfilariae fol- diphosphoglycerates could explain its inhibition lowing diethvlcarbamazinc treatment (Vickel T el of the mutase enzwne. These authors puritied the al., 1986). Thus T cells and T-dependent enzyme and compared some of its properties with ,'esponses (Ig(; and lgE) do not appear to be mamnaalian l~hosphoglyceromutase that were not invoh'ed. The involvenlent of complement also inhibited by clorsuhm. There appears to be no seems unlikeh' because clearing of microfilaria recent studies on the mode of action of this series ii-om the nuc(e mice by diethvlcarbanmzine was of compounds ahhough clinical trials and efficacy not inhibited by prio," treatment with cobra studies have been made. venom lactor treatment (Vicke D' el al.. 1986). Fffects on arachidonic add metabolism may be the DIETHYLCARBAMAZINE mode of attire1 of diellg, lcarbamazine The cllelnical structure of diethylcarbamazine a Diethylcarbanlazine has an antagonistic action piperazine derivative is shown in Fig. 17. Elect,-o- of the metabolic enzymes that ,netabolize arachi- physiological experiments following bath appli- donic acid, tile products released as a resuh of cation to A. suum of high concentrations of phospholipase A.., action on cell membranes. Fig. diethvlcarbamazine have shown that it does not 19 illustrates the sites of action of diethvlcarbama- mimic the action of piperazine (Martin, 1985). zine proposed by Maizels and Denham (1992). Ahhough 5-1ipoxygenase is often being cited as Effects of diethylcarbamazine being inhibited by dietlaylcarbamazine, tiffs may A major indication for diethylcarbamazine at not be the case. In a mast cell line, conversion of low doses is as an anti-filarial drug; it is a good 5-HPETE to leukotriene (LT),&~ by I.TA~ svnthet- microfilaricide (including microfilariae of the dog ase was blocked by diethylcarbamazine hearnvorm, Dirofilana immilis) but has limited (Mathews & Murphy, 1982). Razin el aL (1984) macrofilaricidal effects. The limited action of found that in mouse, 5-1ipoxygenase conversion of diethylcarbamazine against some aduh hehninths arachidonic acid to 5-HPETE was not inhibited (e.g. cattle lung3vorm, Dicl~,ocauhts) requires but that LT(h and LTA_~ production was inhibited. nearly 10 times the dose of the prophylactic anti- Diethylcarbanlazine also appears (Kanesa- filarial dose, and may there£bre, invoh,e another Thasan el al., 1991) to inhibit endothelial pro- mode of action. This review covers the mode of duction by cyclo-oxygenase of prostaglandin action of low closes of diethylcarbamazine; little is (PG)I._, (prostacyclin) and PGE., production but known of the mode of action of high doses of does not affect platelet production of thrombox- diethylcarbamazine. ane (TX)A._, by cyclo-oxygenase and the 12-1ipoxy- Most studies suggest that diethylcarbamazine genase products 12- ancl 15-HPETE. Interestingly, has no direct action on filarial parasites (Johnson microfilariae also produce their own PGI._, and el al., 1991). It appears to have no action at PGE,, and like endothelial cell of blood vessels, reasonable concentrations in vilro. In marked con- diethylcarbamazine also inhibits the microfilarial trast to in vitro experiments, it is known that in production (Kanesa-Thasan el al., 1991). The co- vivo diethylcarbamazine is effective within 4 rain administration of glucocorticoids, that are known following intravenous injection (Hawking & to block phospholipase A,_, activity and the pro- Laurie, 1949). The specific immune response duction of arachidonic acid (Fig. 19), together M()DES OF :M71"ION OF ANTHEI.MINTIC DRL'(;S 31 MEMBRANE PHOSPHOLIPIDS SE 1 PLA 2 ..... 1~- GLUCOCORTICOIDS ...... 1 r ...... i i i DIETHYLCARBAMAZINE i i...... "--r ...... ARACHIDONIC , DIETHYLCARBAMAZINE ACID .-"-" -1 1_ _ andNSAIDS 5-LIPOXYGENASE /J ~_, CYCLO-OXYGENASE / i """"'~ PGGo and 5-HPTE PGD~ PG-H+ '~.~"". • mast-cells 5-HETE 12- and 15- / LIPOXYGENASE \~ / LTA I SYNTHASE / , ~ PGE2 \\ •.\ macrophages; microfilaria 12 and 15 LTA4 HPTE/HETE '\ PGI.,: PROSTACYCLIN \ \ platelets; neutrophils; \ vascular endothelium and eosinopils. \.. microfilaria LTC 4 SYNTHASE \ \ LTC.I LTB4 TXA2: THROMBOXAN eosinophil; neutrol~hils; platelets mast cell macropnages LTD4 eosinophil; mast cell I LTE 4 eosinophil; mast cell Fig. 19. Diagram of the pr(~duction of the leukotrienes and prostaglandins via lipoxygenase or cyclo-oxygenase fi-om ar,tchidonic acid thal is produced from membrane phospholipids as a result ol phospholipase A=, acidity. The inhibitory silc.s of action of gluccwo,ticoids, non-steroidal anti-inllammatola' drugs (NSAIDs) and diethvlcarbamazine are shown. l)iethvlcmbamazine is shown inhil)iting the action of LTA, svnthase thal converts 5-hydr,>peroxyeicosatetraenoic acids (5- HPE'I:E) and 5-1avdroxveicosatet,aenoic acids (5-HETE) to the let,kotrienes I.TAa and I+TA~ to LTC; in mast cells and eosinophils. Dietl~vlcarbamazine is also shown inhibiting the cych>-oxygenase enzyme that converts a,achidonic acid to the prostaglm]dins P(~G._, and P(;H._,. The subsequent production of P(;D_,. PGE=,, PGI=, & TY~-k,_, is also inhibited in the blood vessels and ,nicrol]lari;~. with diethylcarbamazine, reduces the effect of filariae and cytotoxic activig, by the host platelets dietlaylcarhamazine (Stingl el al., 1988). Thus, and granulocytes. It appears that diethylcarbamaz- diethylcarbmnazine seems to inhibit production ine activates an innate immune response rather of PGI=, and PGE=, hy microfilaria and endothelial than an adaptive immune response (Maizels & cells. The normal level of production of PG1._, and Denham, 1992). This mode of action can explain PGE,_, that produces a tonic dilation of blood ves- why diethylcarbamazine has no action in vitro sels and inhibits aggregation of neutroplails and against microfilariae and is effective in non- eosinophils is reduced by administration of immune animals. diethylcarhamazine. A plausible explanation of the mode of action of diethylcarbamazine is that this compound alters the metabolism of arachidonic acids in host endo- ACKNOWLEDGEMENTS thelial cell and also in microfilariae that are sus- ceptible to the action of diethylcarbamazine. 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